LPAR1 regulates enteric nervous system function through glial signaling and contributes to chronic intestinal pseudo-obstruction

Gastrointestinal motility disorders involve alterations to the structure and/or function of the enteric nervous system (ENS) but the causal mechanisms remain unresolved in most cases. Homeostasis and disease in the ENS are processes that are regulated by enteric glia. Signaling mediated through type I lysophosphatidic acid receptors (LPAR1) has recently emerged as an important mechanism that contributes to disease, in part, through effects on peripheral glial survival and function. Enteric glia express LPAR1 but its role in ENS function and motility disorders is unknown. We used a combination of genetic, immunohistochemical, calcium imaging, and in vivo pharmacological approaches to investigate the role of LPAR1 in enteric glia. LPAR1 was enriched in enteric glia in mice and humans and LPA stimulated intracellular calcium responses in enteric glia, subsequently recruiting activity in a subpopulation of myenteric neurons. Blocking LPAR1 in vivo with AM966 attenuated gastrointestinal motility in mice and produced marked enteric neuro- and gliopathy. Samples from humans with chronic intestinal pseudo-obstruction (CIPO), a severe motility disorder, showed reduced glial LPAR1 expression in the colon and ileum. These data suggest that enteric glial LPAR1 signaling regulates gastrointestinal motility through enteric glia and could contribute to severe motility disorders in humans such as CIPO.


Human sample collection
Full thickness colon (n=6) or ileum (n=5) (one patient had both ileum and colon) tissue was obtained from adults (n=10; 8 females; age range: 22-73 years) with a diagnosis of CIPO Comparable tissue samples (n=4 ileum; n=4 colon), obtained from n=4 patients (2 females; age range: 48-73 years) (asymptomatic for previous GI symptoms and referred to surgery for non-complicated GI tumors) were used as controls.

Clinical features of patients
Patients with CIPO secondary to infectious, neurological, metabolic, systemic autoimmune, and paraneoplastic conditions as well as cases secondary to known genetic abnormalities were excluded. Thus, all patients included in this study were idiopathic in origin.
Anorexia nervosa was also specifically excluded in all patients with malnutrition by a psychiatric examination.
The clinical diagnosis of CIPO was established on the basis of a chronic (>3 months), severe symptom complex mimicking mechanical small bowel obstruction, and (at least on one occasion) radiological evidence of air-fluid levels or dilated small bowel loops.
Prior to laparoscopic surgery for tissue collection, patients completed a clinical questionnaire reporting the following data, symptoms, and signs: age, sex, BMI, onset of symptoms, number of sub-obstructive episodes that occurred prior to surgery, abdominal distension, abdominal pain, nausea, vomiting, fullness, early satiety, constipation, diarrhea, esophageal involvement (i.e. motor abnormalities assessed by standard esophageal manometry), gastroparesis (established by scintigraphic gastric emptying), small intestine bacterial overgrowth (SIBO) (determined by glucose/lactulose breath test), urinary symptoms, and small bowel dilation (detectable at abdominal CT scan or MRI). Medications used by each subject were also recorded.
These features were summarized in Table 1.
In 4 patients with CIPO, the ileum was the main site of the disease with at least 3/4 patients reporting symptoms of nausea, vomiting, abdominal fullness, early satiety, diarrhea, or constipation. Of these ileum biopsies, only one derived from a male patient while the remaining three were from female patients. In a separate group of 6 patients with CIPO, the disease was localized to the colon. All patients with colonic CIPO reported experiencing constipation while diarrhea was only reported by one patient. In addition, 4 patients with colonic CIPO reported experiencing mild-to-moderate grade abdominal pain while 1 patient reported experiencing severe abdominal pain. Importantly, the body mass index (BMI) of patients with CIPO affecting the ileum (16.28±1.18, n=4) was lower than that of patients where CIPO affected the colon (20.40±0.39, n=6; p=0.033) suggesting differences in long-term nutritional status between the two groups.

Animal Use
All experiments were conducted according to guidelines established by the National Institutes of Health (NIH) Guide for the Care and Use of Animals. All protocols were subject to rigorous institutional oversight administered by the Michigan State University Institutional Animal Care and Use Committee (IACUC).

Deparaffinization and antigen retrieval
Human tissues were deparaffinized and subjected to heat-induced epitope retrieval using a modified version of techniques described elsewhere(1). Briefly, deparaffinization involved fully submerging slides in two changes of xylene followed by one change of 1:1 xylene:ethanol solution. At this stage, slides were submerged for 10min during each submersion step. Tissues were then hydrated by submerging slides in two changes of 100% ethanol for 3 minutes, 95% ethanol for 1 minute, 90% ethanol for 1 minute and 80% ethanol for 1 minute before being gently rinsed in distilled water. Antigen retrieval was performed by submerging slides for 40 mins in a dish containing sodium citrate buffer solution (10mM sodium citrate, 0.05% Tween-20, pH 6.0), which was preheated on a hotplate to a constant temperature of 95-100 o C. Samples were then removed and allowed to cool to room temperature before conducting immunohistochemical studies.

Animal models
Transgenic Wnt1 Cre ;GCaMP5g-tdT mice generated on a C57/Bl6 background were used to study calcium (Ca 2+ ) responses in the ENS. As described previously, Cre recombinase in Wnt1+ cells of the neural crest drives the expression of the fluorescent Ca 2+ sensor, GCamp5g, in both neurons and glia while the tdTomato reporter is only expressed in glia. This allows Ca 2+ responses to be readily distinguished between these two cell populations. For all experiments, male and female animals were used between 10 and 14 wks of age and were housed in a temperature-regulated facility where they were exposed to a 12 hr light:dark cycle and provided ad libitum access to standard chow and water. Mouse genotypes were verified commercially by Transnetyx Inc. (Cordova, TN). For immunohistochemistry and motility-related experiments, WT C57/Bl6 mice were used.

Mouse colonic tissue isolation and processing
Mice were euthanized by cervical dislocation followed by decapitation in accordance with guidelines established by the Michigan State University institutional ethics policy. Fresh colon tissue was collected in cold Dulbecco's Modified Eagle Medium (DMEM) before being further processed for use in Ca 2+ imaging, immunohistochemistry, or colonic migrating motor complex (CMMC) studies. For Ca 2+ imaging studies only, the DMEM solution was supplemented with 3 µM nicardipine to paralyze the muscular apparatus and enable recording of ENS responses.

Tissue dissection and fixation
Isolated colonic tissue was longitudinally slit open along the mesenteric border and pinned out using insect pins in a 35 mm dish coated with Sylgard TM . The tissue was then fixed with Zamboni's fixative overnight at 4 o C for subsequent immunohistochemical studies. 4% paraformaldehyde (4% PFA) was used instead for RNAscope. For Ca 2+ imaging studies, the epithelium was removed and whole-mount circular muscle-myenteric plexus (CMMP) preparations were generated by flipping the tissue and removing the longitudinal muscle layer by microdissection (2). In the resultant preparation, the myenteric plexus maintains its structural integrity as the thick circular muscle layer is not damaged.

Immunohistochemistry
Following overnight fixation in Zamboni's buffer or 4% PFA, mouse colonic tissue was further micro-dissected according to techniques described previously. In brief, the epithelium was removed along with the circular smooth muscle layer thereby generating a longitudinal musclemyenteric plexus (LMMP) preparation. At this point, a 1x1 cm segment of tissue was then cut out and washed three times in phosphate buffered saline (PBS) containing 0.1% Triton X-100 (PBST).
For both human and fixed mouse tissues, samples were incubated for 40 mins with a blocking solution containing 4% normal goat serum, 0.4% Triton X-100 and 1% bovine serum albumin dissolved in PBS. Tissues were then incubated overnight at 4 o C with the following antibodies: The specificity of rabbit anti-LPAR1 antibody has been confirmed elsewhere(3) but was further demonstrated here by conducting a pre-adsorption assay between the primary antibody and its immunizing peptide sequence (Cat#10006984, Cayman, Ann Arbor, MI) according to manufacturer instructions. Briefly, this entailed incubating the anti-LPAR1 antibody with the immunizing peptide (GGYLPFRDPNSEENSNDIAL) in a 1:1 ratio (v/v) for 1 h at room temperature with occasional mixing. The antibody-peptide cocktail was then diluted to the usual 1:250 antibody dilution and the remaining protocol proceeded as previously described.

Fluorescence in situ hybridization (RNAscope)
Colonic LMMP tissue for RNAscope was generated following fixation and dissection procedures described above (see sections: tissue dissection and fixation and immunohistochemistry). RNAscope was performed using the Advanced Cell Diagnostics RNAscope 2.5 HD Assay-RED (ACD, Cat#322350) according to manufacturer instructions with adjustments for colonic LMMP tissue. Briefly, tissue was dehydrated and subsequently rehydrated by a serial ethanol gradient (25%, 50%, 75%, 100% in PBST) before H2O2 treatment. Tissue was then digested with Protease III for 45 mins and incubated with probes for Lpar1 (ACD, Cat#318591), DapB (ACD, Cat#310043), Ppib (ACD, Cat#313911), Ret (ACD, Cat#431791) or Sox10 (ACD, Cat#435931) overnight at 40°C. All RNAscope steps were performed in a 96-well plate while wash steps were performed in a 48-well plate for 3x5 mins. Following completion of the RNAscope protocol, immunohistochemistry and tissue mounting were performed as described above.

Star Pro peristaltic pump (World Precision Instruments Inc.). Unless otherwise specified, all
imaging studies were conducted on an upright Olympus BX51WI fixed-stage microscope (Center Valley, PA). Ganglia were viewed at 20x through a wide-field water-immersion objective lens (Olympus XLUMPLFLN20xW, 1.0 numerical aperture). The fluorescent tdTomato reporter was highly expressed in resident glial cells. Illumination for fluorescence imaging was provided by a DG4 Xenon light source (Sutter Instrument, Novato, CA).
GCamp5g photoexcitation was filtered through a 485/20-nm band-pass filter, and emitted light was filtered through a 515-nm long-pass filter. tdT signal was excited by light filtered through a 535/20-nm band-pass filter and reflected tdT signals were filtered through a 610/75-nm bandpass emission filter (4). Imaging data were acquired at a frame rate of 0.2 -1 frames per second using a Neo sCMOS camera (Andor, South Windsor, CT). In some cases, confocal video fluorimetry was performed using a confocal spinning-disk microscope (Nikon A1R HD25; Nikon, Tokyo, Japan), providing higher spatiotemporal resolution of Ca 2+ responses, which were imaged through a 20x Nikon objective lens (CFI Apochromat LWD Lambda20xC WI, 0.95 numerical aperture). Confocal images were captured using a Nikon DS-Ri2 digital camera (Nikon, Tokyo, Japan) and recorded using NIS-Elements C software (Nikon). For epifluorescence and confocal fluorescence imaging studies, all data were saved on a personal computer running Windows 10 (Microsoft Corporation, WA) and MetaMorph (Molecular Devices, Sunnyvale, CA) and exported as .tiff stacks for analysis with Fiji software (NIH) (5). To inhibit glial metabolism, some CMMP whole mounts were incubated with FA (5 mM dissolved in DMEM; 37°C; 5% CO2, 95% air) for 2 h before tissue imaging as previously described.

Electrical field stimulation
Electrical field stimulation (EFS) was used to activate neurons using electricity. This was accomplished by applying depolarizing pulses to the tissue using two platinum wires using a GRASS S9E Electrical Stimulator (Quincy, MA) with the parameters: +70V, 10Hz and 0.1ms duration.

Local drug application
ADP or 18:1 LPA were applied locally to the ganglion surface to provoke agonist responses. To do this, glass capillary applicators were fabricated with a pipette puller (P-87 Flaming-Brown Micropipette Puller, Sutter Instruments Corporation, Novato, CA, USA) and backfilled with drug, dissolved in Kreb's buffer. Drugs were then applied using very gentle positive pressure that was applied with a 1-mL syringe connected to a pipette holder. This approach delivered picoliter volumes of drug and only affected the ganglion within the field of view. We confirmed that the shear fluid stress associated with drug application did not activate neurons or glia under these conditions (not shown).

Data analysis and statistics
All analyses were conducted in Fiji (5). Briefly, video analyses were conducted after background-subtracting recordings, which were first stabilized using the StackReg plugin protein expression in the human and mouse ENS was computed by normalizing cross-sectional protein abundance to total ganglionic area and/or net ganglionic cell count. Grubb's test was used to compute statistical outliers, which were excluded from the final analysis only if the confidence threshold of α=0.05 was surpassed. In most instances, statistical testing involved unpaired twotailed Student's t-tests comparing ΔF/Fo values between different conditions. Welch's correction was applied as needed when data variability was unequal between the groups being compared.

AM966 injections and carmine transit studies
Age-matched males and females were randomly assigned to one of 4 possible treatment groups: low-dose AM966 (22 mg/kg), medium-dose AM966 (33 mg/kg), high-dose AM966 (43 mg/kg) or vehicle (7). WT C57/Bl6 mice were then injected intraperitoneally with the LPAR1 antagonist, AM966 (Cat#22048; Cayman, Ann Arbor, MI) q24 for 3 days, or vehicle. The vehicle control consisted of DMSO dissolved in sunflower oil which at most delivered a DMSO dosage far below the 10 ml/kg upper limit reported for mice (8). Mouse weights were measured longitudinally over the duration of the experiment, beginning on the first day of injections. Drug dosages were selected based on previous in vivo reports of the systemic effects and pharmacokinetic properties of AM966 in mice as well as in vitro studies on the binding affinity of AM966 at the LPA1 receptor.
After 3-days of continuous exposure to AM966, the in vivo effect of sustained, sub-chronic LPAR1 blockade was assessed by determining the whole-bowel transit of Carmine red dye. As described elsewhere (9)(10)(11), mice were administered 200 µL of 6% Carmine red solution in water supplemented with 0.5% methylcellulose by oral gavage. After 1h, mice were euthanized, and the entire GI tract was carefully but rapidly removed and laid out at neutral length on an all-white surface. The Carmine red wavefront was then grossly visualized within the gut lumen as a discrete, bright-red bolus that was immediately trailed by a dye-free bowel segment, and its distance relative to the gastric fundus was measured. Additional parameters that were noted included total bowel length, splenic weight, location of the ileocecal pouch and the presence of strictures or serosal hemorrhages. The colonic regions of these tissue were then collected in drugfree DMEM for subsequent fixation and further immunohistochemical characterization of ENS architecture.

Colonic motor complexes (CMCs)
Colonic motility was assessed in response to LPAR1 receptor activation ex vivo. Briefly, whole intact colon was rapidly collected in drug-free DMEM/F-12 maintained at 37 o C. The oral and aboral ends of the colon were then mounted in place on a stainless-steel holding rod after lumen contents were flushed. Two force transducers (Grass Instruments) were connected to the gut wall via surgical silk approximately 2cm apart. The baseline tension was calibrated to 0.5 g and spontaneous CMC production was monitored for a period of 20 min before experiments were conducted. The last 5 min of this acclimatization period was used as a baseline for analysis, which was conducted in LabChart 8 (ADInstruments, Colorado Springs, CO). Drugs were added cumulatively to the bath and the resultant changes in CMC amplitude and integral were measured in relation to baseline.